兔肝金属硫蛋白β结构域的制备和鉴定

制备一定量的Ｃｄ、Ｚｎ兔肝金属硫蛋白,然后脱掉其中的金属,获得不含金属的硫蛋白,用一价金属ＣｕＣｌ或ＡｇＮＯ３以５．８：１的金属：蛋白摩尔比与ａｐｏＭＴ结合,将其β结构域用金属饱和,在３７℃,ＰＨ７．０下用枯草杆菌蛋白酶水解掉未结合金属的α结构域,获得Ｃｕ（Ⅰ）、Ａｇ（Ⅰ）结合的兔肝ＭＴ－Ⅰ、ＭＴ－Ⅱ的β结构域,通过ＨＰＬＣ、羧甲基化后的ＳＤＳ－ＰＡＧＥ、氨基酸组成分析、金属定量、Ｎ末端分析和紫外     

金属硫蛋白α和 β结构域的结构功能比较研究

金属硫蛋白具有α和β两个独立的结构域,它们结构不同,并能独立的行使功能。为了进一步研究这两个结构域之间的区别,分别采用镉和铜重组金属硫蛋白并继以枯草杆菌蛋白酶水解的方法制备α和β结构域,以及利用pGEX-4T-1这种融合表达载体表达α和β结构域。所得产物经凝胶过滤层析分离纯化后,进行了氨基酸组成,巯基和金属含量以及分子量测定,以上性质均与天然的金属硫蛋白α和β结构域相同。然后利用紫外吸收光谱和圆二色吸收光谱来研究它们的巯基金属簇结构,从UV和CD图谱可以看出,通过蛋白水解和基因表达制备的α和β结构域都具有独立的镉硫金属簇结构,但β结构域的镉硫金属簇不如α结构域紧密,其在254nm的吸收峰不象α结构域那么明显。利用DTNB的竞争反应测定了α和β结构域对镉和锌的结合力,实验结果表明,α结构域倾向于结合Cd2+,β结构域倾向于结合Zn2+。以上研究对于进一步了解α和β结构域的生理功能和分子进化提供了有利的证据。     

兔肝金属硫蛋白β结构域的制备和鉴定

制备一定量的Ｃｄ、Ｚｎ兔肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,ＭＴ）,然后脱掉其中的金属,获得不含金属的硫蛋白（ａｐｏＭＴ）．用一价金属ＣｕＣｌ或ＡｇＮＯ３以５．８１的金属蛋白摩尔比与ａｐｏＭＴ结合,将其β结构域用金属饱和,在３７℃,ｐＨ７．０下用枯草杆菌蛋白酶水解掉未结合金属的α结构域,获得Ｃｕ（Ⅰ）、Ａｇ（Ⅰ）结合的兔肝ＭＴ－Ｉ、ＭＴ－Ⅱ的β结构域．通过ＨＰＬＣ、羧甲基化后的ＳＤＳ－ＰＡＧＥ、氨基酸组成分析、金属定量、Ｎ末端分析和紫外扫描等鉴定,证明确实得到了Ｎ末端为Ｍｅｔ,分子量３０００左右,金属蛋白摩尔比为５～５．５１的ＭＴβ结构域,其氨基酸组成与理论值基本相符     

一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析

从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。     

金属硫蛋白的生理功能及其研究进展

金属硫蛋白的生理功能及其研究进展赵京山尹桂山（河北医科大学生化教研室，石家庄０５００１７）关键词金属硫蛋白生理功能检测方法１９５７年Ｍａｇｏｓｈｅ和Ｖａｌｅｅ在马的肾脏中发现一种富含Ｃｄ、Ｚｎ和半胱氨酸的低分子量蛋白质，随后Ｋａｇｌ和Ｖａｌｅｅ将其提...     

脑金属硫蛋白GIF及其功能研究

脑金属硫蛋白ＧＩＦ及其功能研究王文清,潘宪明,刘杰（北京大学技术物理系１００８７１）（美国Ｋａｎｓａｓ医学中心）近年来在正常人脑皮层中发现了一种新的金属硫蛋白异构体,称ＧＩＦ（Ｇｆ。Ｗｔｈｌｎｈｉｂｉ－ｔｏｒｙＦａｃｔｏｒ,１９９１,Ｙｏｋ。Ｕｃｈｉ...     

无脊椎动物金属硫蛋白的研究

综述了近几十年来有关无脊椎动物金属硫蛋白（MT）的研究,包括软体动物MT、棘皮动物MT、环节动物MT、节肢动物MT（甲壳纲和昆虫纲）,分析了无脊椎动物MT与哺乳动物MT的异同,在此基础上,提出以下观点：即从无脊椎动物MT到哺乳动物MT的进化来看,动物MT的进化可能是一种趋同进化的模式。     

金属硫蛋白

金属硫蛋白为一类广泛地存在于生物界的低分子量、富含半胱氨酸的金属结合蛋白,尤其对Cd,Zn,Cu和Au,Ag等有较强的亲和力。它是一种诱导合成的蛋白质,在体内能被金属和糖皮质激素等所诱导。它具有广泛的生物学功能,主要是参与微量元素的储存、运输和代谢,拮抗电离辐射,清除羟基自由基和重金属解毒等多种作用。某些微量元素缺乏症等疾病也与它有一定关系。今后在某些疾病的治疗、环保中清除有毒金属以及采矿、富集贵重金属等方面有着广泛的应用前景。     

圆二色光谱在蛋白质结构研究中的应用

近几年来,圆二色光谱在蛋白质结构研究中的应用越来越广泛。通过对远紫外圆二色光谱的测量,可以推导出稀溶液中蛋白质的二级结构,进而分析和辨别蛋白质的三级结构类型;通过对近紫外圆二色光谱的测量和分析,可以推断蛋白质分子中芳香氨基酸残基和二硫键的微环境变化,研究介质与蛋白质结构间的关系;通过测定实验参数和环境条件变化时的圆二色光谱,可以研究蛋白质构像变化过程中的热力学和动力学特性。     

心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

Domain structure of flagellin

The chemotaxis of bacteria such as Salmonella and Escherichia coli involves smooth swimming punctuated by periods of tumbling. In smooth swimming the flagellar filaments are left-handed, in tumbling they are right-handed with a different wavelength. The filaments are constructed from a globular protein, flagellin, by a process of self-assembly. The existing models assume that the flagellin molecule is bistable and longitudinal rows of subunits take one of the two possible conformations. Such a model explains the observed different morphology of the flagellum. We have studied Salmonella and E. coli flagellins in polymeric and monomeric forms by scanning microcalorimetry and circular dichroism. We have inferred that a flagellin molecule consists of several domains, two of which are structured at physiological temperatures and are in the monomeric form, while the others acquire a regular form only in the process of polymerization. This phenomenon may be the basis of a process during which the flagellin molecule, fitting into the flagellum, acquires a conformation analogous to that of the neighbouring molecule in the longitudinal row.     

岩豆凝集素分子修饰与圆二色性的关系研究

天然态岩豆凝集素（ＭＤＬ）远紫外圆二色性（ＣＤ）谱显示２１６ｎｍ处单一负峰,是一种高β－折叠构象凝集素;近紫外ＣＤ谱呈现２８２ｎｍ处负峰和２６０～２７５ｎｍ及２９５ｎｍ处的负肩,经Ｎ－乙基顺丁烯二酰亚胺（ＮＥＭＩ）和对氯汞苯甲酸（ＰＣＭＢ）修饰ＭＤＬ的巯基,其近紫外ＣＤ谱未发生变化,远紫外ＣＤ谱仅发生细微变化,ＭＤＬ凝集活性保持不变;ＰＣＭＢ过量时,ＣＤ谱呈现典型的无规卷曲谱形,ＭＤＬ完全丧失凝集活性,去除ＰＣＭＢ后,活性又全部恢复．二硫苏糖醇（ＤＤＴ）修饰ＭＤＬ的二硫键并用碘乙酸（ＩＣＨ２ＣＯＯＨ）保护巯基,ＭＤＬ远紫外ＣＤ谱２１６ｎｍ处的负峰红移至２２５ｎｍ,且显著减小;同时,近紫外ＣＤ谱２８２ｎｍ处负峰几乎消失,两负肩分别保持完整,分子中α－螺旋降低,无规卷曲增加较多,ＭＤＬ凝集活性未发生变化．用Ｎ－溴代丁二酰亚胺（ＮＢＳ）修饰ＭＤＬ分子中的色氨酸,导致２１６ｎｍ负峰蓝移至２０８ｎｍ且变小,分子中无规卷曲和α－螺旋增加,β折叠减少,近紫外ＣＤ谱２９５ｎｍ负肩消失,２８２ｎｍ负峰红移至２８７ｎｍ,ＭＤＬ凝集活性完全丧失．     

皖南尖吻蝮蛇出血毒素生物学活性与圆二色性关系的研究

用远紫外ＣＤ谱研究了皖南尖吻蝮蛇蛇毒３个出血毒素ＡａＨⅠ、ＡａＨⅢ和ＡａＨⅣ的溶液构象,ＡａＨⅠ的α螺旋、β折叠、β转角和无规卷曲的含量分别为２５．８％、１２．７％、２６．８％和３４．７％;ＡａＨⅢ的二级结构含量分别为２３．９％、２０．６％、２３．７％和３１．８％;而ＡａＨⅣ分别为１８．２％、３１．０％、１７．２％和３３．６％。当ｐＨ小于４．０或ｐＨ大于１１．０时,３种出血毒素α螺旋减少而β折叠增多,同时３种出血毒素的酪蛋白水解活性显著下降。ＥＤＴＡ抑制酪蛋白水解活性,表明３种出血毒素均是金属蛋白酶。ＥＤＴＡ、Ｃｕ２＋、Ｚｎ２＋、Ｃａ２＋和Ｍｇ２＋能改变３种出血毒素的二级结构,并影响酪蛋白水解活性。     

Studies on the Circular Dichroism (CD) Spectra of Iron-Molyb-denum Protein from Azotobacter vinelandii Nitrogenase

Since the unsymmetry of metal cluster(P-cluster), which was under different redox states, in FeMo protein of A. vinelandii nitrogenase could be observed by circular dichroism, the numbers of the redox equivalent for the P-cluster was obtained by CD titration of DT-stripped FeMo protein with oxidant, i.e. plotting △δ450nm against the number of equivalent of oxidant added. After exposure to air, at the beginning P-cluster was reversibly oxidized, then followed by irreversible oxygen-damage. Dithiothreitol (DTT) was able to increase the C2H2-reduction activity of FeMo protein, which was not severely damaged by O2, or by other stronger oxidants, but could not change their CD spectra. It seems that the reactivation may be not due to the restoration of the P-cluster to its reducing state.     

Study of Interaction of GM2 Activator Protein with GM2 Using Circular Dichroism and Fluorescence Spectroscopy

GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by -hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized -helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2.     

Vibrational Circular Dichroism Discrimination of Diastereomeric Cedranol Acetates

The reliability of vibrational circular dichroism (VCD) spectroscopy to discriminate four diastereomeric cedranol acetates 1 , 2 , 3 , 4 by means of their absolute configuration is examined. The usage of CompareVOA software to quantify comparisons of the measured infrared (IR) and VCD spectra with the corresponding simulated spectra at the B3LYP/DGDZVP and B3PW91/DGDZVP levels of theory for each diastereomer enabled the B3PW91 functional to be qualified as superior to the B3LYP functional for vibrational calculations of 1 , 2 , 3 , 4 . Analogously, a set of quantitative VCD spectra cross‐comparisons of 1 , 2 , 3 , 4 unambiguously distinguished the diastereomers using B3PW91 and failed using B3LYP. Remarkably, quantitative IR spectra cross‐comparisons of 1 , 2 , 3 , 4 using B3PW91 or B3LYP functionals demonstrated that the achiral spectroscopic IR technique is not able to distinguish cedranol acetate diastereomers. VCD comparisons using anisotropy g‐factor values of bands in the 1550–950 cm^‐1 region of the spectra were of aid to facilitate visual spectra matching for each diastereomer. Chirality 25:939‐951, 2013. © 2013 Wiley Periodicals, Inc.     

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外ＣＤ谱研究了湖南产尖吻蝮蛇毒的两个出血毒素（ＤａＨＴ－１、ＤａＨＴ－２）的溶液构象,计算得ＤａＨＴ－１的α螺旋、β折叠、无规卷曲的含量分别为３６．９％、３５．５％、２７．６％;ＤａＨＴ－２的α螺旋、β折叠、无规卷曲分别为２３．４％、３１．３％、４５．３％。随ｐＨ的增大或减小,峰位蓝移,酸性条件下的变化比碱性条件下的变化大。构象单元含量计算表明：α螺旋减少,无规卷曲增多,β折叠基本未变。温度和ｐＨ对ＣＤ谱的影响相似,５０℃时峰位蓝移,α螺旋减少,无规卷曲增多．ＥＤＴＡ对ＣＤ谱影响显著,０．０２ｍｏｌ／ＬＥＤＴＡ便导致两个出血毒素呈极度的无序状态。ＥＤＴＡ完全抑制,半胱氨酸部分抑制,胰蛋白酶不影响它们的出血活性。     

The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments

The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein-coupled receptor adopts a characteristic alpha-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the alpha-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP-HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.     

黄曲霉毒素解毒酶在大肠杆菌中的可溶性表达、纯化及其圆二色谱分析

目的:应用原核表达系统对黄曲霉毒素解毒酶(aflatoxin-detoxifizyme,ADTZ)进行高效可溶表达和纯化,并对其进行生物学活性与二级结构分析。方法与结果:亚克隆ADTZ的成熟肽,并构建与pMAL-c2x的重组质粒,转化大肠杆菌Rosetta(DE3),IPTG诱导实现了MBP_ADTZ融合蛋白的高效可溶表达,其表达量约占总蛋白的50%。经Amylose亲和层析、Factor Xa酶切和疏水层析后得到高纯度的rADTZ蛋白。生物学活性分析表明rADTZ蛋白具有降解AFB₁的酶活性,酶比活为136U/mg。圆二色光谱对rADTZ蛋白二级结构的分析结果为:α-螺旋为43.3%、β-折叠为31.1%、β-转角为10.5%和无规则卷曲为15.1%。结论:用大肠杆菌成功表达并得到高纯度有活性的rADTZ蛋白,为进一步对rADTZ结构与功能研究奠定了基础。